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92 Cards in this Set

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Test for Motility
***This is NOT a biochemical test but must be determined for the Unknowns!
Used to determine whether or not an organism is motile
Medium used for Motility
Motility Medium
Procedure for Motility Test
1 use a needle to inoculate by making a single stab, about 2/3 down and then pull out the needle along the same path. 2 Incubate at the optimum temperature for 24-48 hours.
Interpretation for Motility Test
Motile--the tube will appear cloudly and usually the organism will spread over the top of the media.
Non-Motile--the organism will grow along the streak line only; the media will NOT be cloudy.
Test for Oxygen Requirements
Used for determination of oxygen requirements
Medium used for oxygen requirements
Fluid Thioglycollate Medium
Procedure for Oxygen requirements test
1 Boil and cool media with screw cap loose 2 Inoculate media with the organism using a wire loop. Do not shake the media. 3 Incubate at optimum temperature for 24 hrs.
Interpretation for oxygen requirements Test
Aerobe-growth at the top of the media. Facultative-growth throughout the media Anaerobe-growth at the bottom of the media
Theory for Oxygen requirements test
The media contains glucose, cystine, and sodium thioglycollate to lower the oxidation-reduction potential. The oxygen tension is high at the surface of the media (allowing aerobes to grow) and decreases toward the bottom of the media (for anaerobic growth). Resazurin (a dye) causes the media to turn pink in the presence of oxygen.
Gelatin Liquefaction Test
Used to determine the ability of an organism to produce the enzyme gelatinase, which liquifies gelatin
Medium used for Gelatin Liquefaction Test
Nutrient Gelatin
Procedure for Gelatin Liquefaction Test
1 Stab gelatin with organism using a straight wire 2 Incubate at optimum temperature for 24-48 hours 3 Place tubes in ice water bath for at least 30 minutes
Interpretation for Gelatin Liquefaction Test
Postive--gelatin is liquefied (compare patterns with figure 47.3 in lab manual) Negative--gelatin is solid (Note: continue incubation of negative tubes for another 4 or 5 days to see if gelatinase is produced slowly.)
Theory for Gelatin Liquefaction Test
Gelatinase breaks down large proteins into smaller components, which can then enter the organism and be metabolized. Protein + H2O-->Polypeptides. Polypeptides + H2O --> Amino Acids
Carbohydrate Fermentation
Used to determine the ability of an organism to ferment a specific carbohydrate with or without the production of gas
Medium used for carbohydrate fermentation test
Glucose, Lactose, Mannitol, Maltose and Sucrose broth
Procedure for carbohydrate fermentation test
1 Inoculate each media with the organism 2 Incubate at the optimum termperature for 24-48 hours
Interpretation for Carbohydrate Fermemtation test
Positive- media turns yellow (fermentation has occurred) Negative- media remains red (no fermentation) (NOTES: continue incubation of negative tubes for up to 2 weeks to detet "slow" fermenters. Compare Lactose Fermentation results with Litmus Milk Test.)
Methyl Red Test
Mixed Acid Fermentation. Used to determine the ability of an organism to produce mixed acid end products from glucose fermentation
Medium used for Methyl Red Test
MR-VP Broth
Procedure for Methyl Red Test
1 Inoculate two MR-VP broths with the organism 2 Incubate at optimum temperature for 3-5 days. 3 Add 3-4 drops Methyl Red reagent to one tube.
Theory for Methyl Red Test
Some organisms produce a large amounts of various acids (lactic, acetic, succinic, formic) plus H2 and CO2. The large amounts of acids lower the pH to less than 5.0 These organisms also produce great amounts of gas due to the presence of the enzyme formic hydrogen lyase. Formic Acid--->CO2+ H2
Interpretation for Methyl Red Test
Positive- Red color develops Negative- yellow color develops
Voges-Proskauer Test
Butanediol Fermentation. Used to determine the ability of an organism to produce acetoin (acetylmethyl carbinol), 2,3 butanediol, and ethanoil which cause less lowering of the pH than methyl red positive organisms.
Procedure for Voges-Proskauer test
Using one of the two incubated MR-VP broth from the methyl red ...4 Pipet 1 ml of culture from the other MR-VP tube into a small screw cap test tube. 5 To the extracted 1 ml of culture, add 18 drops Barritt's Solution A (alphanapthol) and 18 drops Barritt's solution B (KOH). 6 Agitate vigorously for 1-2 minutes. Let stand for 1-2 hours.
Interpretation for Voges-Proskauer test
Positive- wine red (burgundy) color develops Negative- brown color develops
Catalase Test
Used to test for presence of enzyme catalase
Medium for Catalase Test
Nutrient Agar Slant
Theory for Voges-Proskauer Test
VP test detects the presence of acetoin, which is a precursor to 2,3 butanediol.
Theory for Catalase Test
Hydrogen peroxide (H2O2) is formed as an end product of the aerobic breakdown of sugars. When H2O2 accumulates, it becomes toxic to the organism. Catalase decomposes H2O2 and enables the organism to survive. Only obligate anaerobes lack this enzyme. 2H2O catalase---->2H2O + O2
Interpretation for Catalase Test
Positive-bubbling (O2 gas is liberated from the H2O2) Negative no bubbling
Procedure for Catalase Test
1 Streak nutrient agar slant with the organism. 2 Incubate at optimum temperature for 24-48 hours 3 Place a few drops of 3% H2O2 on the slant culture.
Oxidase Test
Used to determine the presence of oxidase enzymes
Medium for Oxidase Test
Tryptic Soy Agar Plate
Procedure for Oxidase Test
1 make an isolation streak of the organism on TSA plate 2 Incubate at the optimum temperature for 24-48 hours 4 touch a sterile swab to colonies on a plate 5 add several drops of Oxidase test reagent directly to organism on swab 6 Wait 10-30 seconds.
Theory for Oxidase Test
Aerobic organisms obtain their energy by respiration, which is responsible for the oxidation of various substrates through the cytochrome oxidase system. Obligate aerobes have this enzyme.
Interpretation for Oxidase Test
Positive- organism will change color from yellow to purple ( note: changes after 30 seconds are considered negative reactions) Negative- no color change
Nitrate Reductase Test
Used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or nitrogen gas (N2) by production of the enzyme, nitratase.
Procedure for Nitrate Reductase Test
1 Boil and Cool Media with screw cap loose. 2 Inoculate nitrate broth with the organism 3 Incubate at the optimum temperature for 24-48 hours. 4 Add 5 drops of Nitrate Reagent A (sulfanilic acid) and 5 drops of Nitrate Reagent B (dimethyl alpha naphthalamine) to the tube.
Theory for Nitrate Reductase Test
The reduction of nitrate to nitrite or nitrogen gas takes place under anaerobic conditions in which an oganism derives its oxygen from nitrate. Nitrate (NO3) Nitratase----> Nitrite (NO2) -----> N2 + NH3
Interpretation for Nitrate Reductase Test
Positve- red color; nitrate reduced to nitrite; test is complete Negative- no color change; do confirmation Test by adding a small pinch of zinc powder. Interpretation of confirmation test: Positive- no color change; organism reduced nitrate completely to ammonia and nitrogen gas. Negative--Red color; nitrate reduced by zinc, not the organism (confirms first negative)
Medium for Nitrate Reductase Test
Nitrate Broth
Starch Hydrolysis
Used to determine the ability of an organism to hydrolyze (break down) starch.
Procedure for Starch Hydrolysis
1 Make a single streak of the organism on a starch agar plate 2 Incubate at the optimum temperature for 24-48 hours. 3 Drop a small amount of IKI (Gran's Iodine) onto the plate and rotate the plate gently. (iodine is an indicator of starch; in the presence of starch the iodine will turn blue/black)
Theory for Starch Hydrolysis
The enzyme amylase breaks starch down into components more easily metabolized by the organism. Starch----> Maltose+Glucose+Dextrin
Medium for Starch Hydrolysis
Starch Agar Plate
Interpretation for Starch Hydrolysis
Positve- a zone of clearing appears adjacent to the streak line Negative no clearing; only a blue/black area surrounding the streak line.
Casein Hydrolysis
Used to determine the ability of an organism to produce the enzyme caseinase which hydrolyzeds (breaks down) casein ( a white protein in milk) to more soluble products
Procedure for Casein Hydrolysis
1 Make a single streak of the organism on a skim milk agar plate. 2 Incubate at the optimum temperature for 24-48 hours
Medium for Casein Hydrolysis
Skim Milk Agar
Interpretation for Casein Hydrolysis
Positive- a zone of clearing occurs along the streak line. Negative-no zone of clearing (NOTE: compare results with the Litmus Milk Test)
Fat Hydrolysis
Used to determine the ability of an organism to produce the enzyme lipase which hydrolyzes (breaks down) fat.
Theory for Fat Hydrolysis
Lipase splits fats into glycerol and fatty acids that can be used to anabolism or energy production Triglycerides (fats)-----> Glycerol + 3 fatty acids. The medium contains vegetable oil that, when hydrolyzed, lowers the pH
Medium used for Fat Hydrolysis
Spirit Blue Agar Plate
Procedure for Fat Hydrolysis
1 Make a single streak of the organism on a spirit blue agar plate. 2 incubate at optimum temperature for 24-48 hours.
Interpretation for Fat Hydrolysis
Positve--a dark blue precipitate forms along the steak line or oil droplets are depleted in this area if the pH is not lowered sufficiently (hold plate up to light to see depletion) Negative--no change in medium
Tryptophan Hydrolysis/ "Indole Test"
Used to determine the ability of an organism to split indole from the amino acid tryptophan using the enzyme tryptophanase. Tryptophan-----> Indole + pyruvic Acid
Hydrogen Sulfide Production
Used to determine the abiliy of an organism to produce H2S
Medium used for Tryptophan Hydrolysis/ "Indole Test"
Trypotone Broth
Procedure for Tryptophan Hydrolysis/ "Indole Test"
1 inoculate brothe with organism 2 Incubate at optimum temperature for 24-48 hours 3 Add 10-12 drops of Kovacs Reagent
Interpretation for Tryptophan Hydrolysis/ "Indole Test"
Positive-red layer forms on surface of media Negative--yellow layer on surface of media
Urease Test
Used to determine the ability of an organism to split urea to form ammonia (an alkaline end product) by the action of the enzyme, urease. Media also contains the pH indicator phenol red, which turns an intense pink at alkaline pH. Urea----> 2 ammonia + CO2
Medium used Hydrogen Sulfide Production
Kligler's Iron Agar
Medium used Urease Test
Urea Broth
Procedure for Urease Test
1 Inoculate urea broth with the organism 2 incubate at optimum temperature for 24-48 hours
Interpretation for Urease Test
positive--intense pink/red color Negative--no color change (NOTE: continue incubation of negative tubes for a total of 7 days to check for slow urease producers)
Theory for Hydrogen Sulfide Production
Medium also contains glucose and lactose and phenol red as a pH indicator to show fermentation of these sugars. Gaps, cracks, or bubbles in agar indicate gas production.
Procedure for Hydrogen Sulfide Production
1 Stab KIA with straight wire inoculum or organism. 2 Incubate at optimum temperature for 24-48 hours
Interpretation of Hydrogen Sulfide Production
Positive-black precipitate along stab line Negative--no precipitate
Citrate Utilization
Used to determine if an organism is capable of using citrate as the sole source of carbon with production of enzyme citratase.
Theory for Citrate Utilization
The media contains sodium citrate as the carbon source, and ammonium salts as the nitrogen source, with bronthymol blue as the pH indicator. An organism that uses citrate breaks down the ammonium salts to ammonia, which creates an alkaline pH.
Procedure for Citrate Utilization
1 Stab/Streak Simmons citrate agar slant with the organism 2 incubate at the optimum temperature for 24-48 hours
Medium used for Citrate Utilization
Simmons Citrate Agar
Interpretation of Citrate Utilization
Positive- alkaline pH causes media to change from green to prussian blue Negative- no color change
Phenylalanine Deamination
Used to determine the abiliy of an organism to deaminate the amino acid phenylalanine resulting in the production of phenylpyruvic acid and ammonia. This reaction is catalyzed by the enzyme phenylalanase.
Theory for Litmus Milk test
Litmus is used as a pH and oxidation-reduction indicator. In un-inoculated milk, litmus will be purple/blue (pH 6.8). In acid solution (pH 4.5) litmus will be pink, and in alkaline solution (pH 8.3) litmus will be blue.
Procedure for Phenylalanine Deamination
1 Steak phenylalanine agar slant with the organism 2 Incubate at the optimum temperature for 24-48 hours 3 Place 5-10 drops of 10% Ferric chloride on the slant culture. Use a loop to mix organism into solution
Medium used for Phenylalanine Deamination
Phenylalanine Agar
Interpretation of Phenylalanine Deamination
Positive--a deep green color appears within 1-5 minutes Negaive --an amber color develops
Litmus Milk Test
Used to differentiate organisms in skim milk agar according to metabolic properties: 1 lactose fermentation 2 Reduction of litmus 3 Clot formation 4 Peptonization (digestion)
Medium used for Litmus Milk test
Litmus milk tube
Lactose Fermentation in Litmus Milk Test
If the organism can ferment lactose, an acid condition occurs and the media will be pink. If the organism cannot ferment lactose, it may act on nitrogenous substances in the milk to release ammonia and the media will be blue
Reduction of Litmus in Litmus Milk Test
Litmus is used as an oxidation-reduction(Eh) indicator. An organism capable of reducing litmus will cause the media to turn white
Clot Formation in Litmus Milk Test
Proteolytic enzymes (rennin, pepsin or chymotrypsin) cause the hydrolysis of milk proteins, which results in the coagulation of milk.
Peptonization (digestion ) in Litmus Milk Test
Hydrolysis of casein by caseinase causes the caseinogen precipitate (clot) to be converted to a clear fluid.
Procedure for Litmus Milk Test
1 inoculate the litmus milk media with the organism using a wire loop 2 incubate at the optimum temperature for up to 5 days
Interpretation for Litmus Milk Test
Your results will be one or more of the following: Pink: acid reaction, lactose fermented.(verify with carbohydrate fermentation test) Purple/blue: no fermentation of lactose Blue: alkaline reaction, no fermentation of lactose; organism attacks nitrogenous substances. White: reduction of litmus Clearing of media: peptonization (verify with casein Hydrolysis test) Clot/Curd: milk protein coagulation
Lysine Decarboxylase Test
Used to determine the ability of an organism to decarboxylate the amino acid lysine, resulting in the production of the alkaline end-product cadaverine, by producing the enzyme lysine decarboxylase
Theory for Lysine Decarboxylase Test
The media contains lysine, glucose (as a substrate for fermentation), and the pH indicator Brom Cresol purple (purple at alkaline Ph; Yellow at Acid pH). The enzyme requires an acid pH (below 5) for activation. IF the organism is capable of glucose fermentation (check carbohydrate broth), AND has the enzyme lysine decarboxylase, the following events occur: 1 microbe ferments glucose, producing a low pH; indicator turns yellow. 2 Lysine decarboxylase is activated. 3 Cadaverine is formed, pH rises and the media returns to its original purple color.
Medium used for Lysine Decarboxylase Test
Lysine Decarboxylase Broth
Procedure for Lysine Decarboxylase Test
1 Inoculate broth with the organism using a wire loop 2 Incubate at optimum temperature for 24-48 hours
Interpretation of Lysine Decarboxylase Test
Positive- purple (verify organism ferments glucose by checking the Carbohydrate Fermentation Test, if the organism does NOT ferment glucose than a purple color is negative. ) Negative--yellow